RESUMO
Multiple sclerosis (MS) and its animal models are characterized by cellular inflammation within the central nervous system (CNS). The sources and consequences of this inflammation are currently not completely understood. Critical signs and mediators of CNS inflammation are reactive oxygen species (ROS) that promote inflammation. ROS originate from a variety of redox-reactive enzymes, one class of which catalyses oxidative protein folding within the endoplasmic reticulum (ER). Here, the unfolded protein response and other signalling mechanisms maintain a balance between ROS producers such as ER oxidoreductin 1α (Ero1α) and antioxidants such as glutathione peroxidase 8 (GPx8). The role of ROS production within the ER has so far not been examined in the context of MS. In this manuscript, we examined how components of the ER redox network change upon MS and experimental autoimmune encephalomyelitis (EAE). We found that unlike GPx8, Ero1α increases within both MS and EAE astrocytes, in parallel with an imbalance of other oxidases such of GPx7, and that no change was observed within neurons. This imbalance of ER redox enzymes can reduce the lifespan of astrocytes, while neurons are not affected. Therefore, Ero1α induction makes astrocytes vulnerable to oxidative stress in the MS and EAE pathologies.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Glutationa Peroxidase/metabolismo , Inflamação , Espécies Reativas de Oxigênio/metabolismoRESUMO
How homeostatic ER calcium fluxes shape cellular calcium signals is still poorly understood. Here we used dual-color calcium imaging (ER-cytosol) and transcriptome analysis to link candidates of the calcium toolkit of astrocytes with homeostatic calcium signals. We found molecular and pharmacological evidence that P/Q-type channel Cacna1a contributes to depolarization-dependent calcium entry in astrocytes. For stimulated ER calcium release, the cells express the phospholipase Cb3, IP3 receptors Itpr1 and Itpr2, but no ryanodine receptors (Ryr1-3). After IP3-induced calcium release, Stim1/2 - Orai1/2/3 most likely mediate SOCE. The Serca2 (Atp2a2) is the candidate for refilling of the ER calcium store. The cells highly express adenosine receptor Adora1a for IP3-induced calcium release. Accordingly, adenosine induces fast ER calcium release and subsequent ER calcium oscillations. After stimulation, calcium refilling of the ER depends on extracellular calcium. In response to SOCE, astrocytes show calcium-induced calcium release, notably even after ER calcium was depleted by extracellular calcium removal in unstimulated cells. In contrast, spontaneous ER-cytosol calcium oscillations were not fully dependent on extracellular calcium, as ER calcium oscillations could persist over minutes in calcium-free solution. Additionally, cell-autonomous calcium oscillations show a second-long spatial and temporal delay in the signal dynamics of ER and cytosolic calcium. Our data reveal a rather strong contribution of homeostatic calcium fluxes in shaping IP3-induced and calcium-induced calcium release as well as spatiotemporal components of intracellular calcium oscillations.
Assuntos
Sinalização do Cálcio , Cálcio , Astrócitos/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Homeostase , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca2+ -sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Estresse do Retículo Endoplasmático , Gânglios Espinais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Neuralgia/metabolismo , Nociceptores/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Gânglios Espinais/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Países Baixos , Nociceptores/patologiaRESUMO
We have shown that multiple sclerosis (MS) and endoplasmic reticulum (ER) stress induce Rab32, an ER/mitochondria-localized small GTPase. High levels of both dominant-active (Q85L) or dominant-inactive (T39N) Rab32 are toxic to neurons. While Rab32Q85L interacts with its effector Drp1 to promote mitochondria fission, it is unclear how Rab32T39N could result as toxic to neurons. Given the perinuclear clustering of mitochondria observed upon transfection of inactive Rab32, we hypothesized Rab32T39N could stall mitochondria within neurites. The movement of mitochondria depends on kinesin-binding Miro proteins. High cytosolic [Ca2+] is bound by an EF hand motif within Miro proteins, resulting in mitochondrial arrest. Consistent with increased cytosolic [Ca2+], expression of Rab32T39N arrests mitochondria movement within neurites.
Assuntos
Mitocôndrias/metabolismo , Neuritos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Citosol/química , Citosol/metabolismo , Humanos , Mutação , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The endoplasmic reticulum (ER) acts as a dynamic calcium store and is involved in the generation of specific patterns of calcium signals in neurons. Calcium is mobilized from the ER store by multiple signaling cascades, and neuronal activity is known to regulate ER calcium levels. We asked how neurons regulate ER calcium levels in the resting state. Direct ER calcium imaging showed that ER calcium was lost quite rapidly from the somatic and dendritic ER when resting neurons were transiently kept under calcium-free conditions. Interestingly, free ER and free cytosolic calcium was lost continuously across the plasma membrane and was not held back in the cytosol, implying the presence of a prominent calcium influx mechanism to maintain ER calcium levels at rest. When neurons were treated acutely with inhibitors of store-operated calcium entry (SOCE), an immediate decline in ER calcium levels was observed. This continuous SOCE-like calcium entry did not require the activation of a signaling cascade, but was rather a steady-state phenomenon. The SOCE-like mechanism maintains medium-high ER calcium levels at rest and is essential for balanced resting calcium levels in the ER and cytosol.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carboxilesterase , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Imidazóis/farmacologia , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Confocal , Proteína ORAI1 , Proteína ORAI2 , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.
Assuntos
Cálcio/análise , Carboxilesterase/química , Retículo Endoplasmático/química , Imagem Óptica/métodos , Animais , Cálcio/metabolismo , Carboxilesterase/biossíntese , Carboxilesterase/genética , Córtex Cerebral/citologia , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Hipocampo/citologia , Humanos , Lentivirus/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Neuroglia/química , Neuroglia/metabolismo , Neurônios/química , Neurônios/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Sulfated glycosaminoglycan chains are known for their regulatory functions during neural development and regeneration. However, it is still unknown whether the sulfate residues alone influence, for example, neural precursor cell behavior or whether they act in concert with the sugar backbone. Here, we provide evidence that the unique 473HD-epitope, a representative chondroitin sulfate, is expressed by spinal cord neural precursor cells in vivo and in vitro, suggesting a potential function of sulfated glycosaminoglycans for spinal cord development. RESULTS: Thus, we applied the widely used sulfation inhibitor sodium chlorate to analyze the importance of normal sulfation levels for spinal cord neural precursor cell biology in vitro. Addition of sodium chlorate to spinal cord neural precursor cell cultures affected cell cycle progression accompanied by changed extracellular signal-regulated kinase 1 or 2 activation levels. This resulted in a higher percentage of neurons already under proliferative conditions. In contrast, the relative number of glial cells was largely unaffected. Strikingly, both morphological and electrophysiological characterization of neural precursor cell-derived neurons demonstrated an attenuated neuronal maturation in the presence of sodium chlorate, including a disturbed neuronal polarization. CONCLUSIONS: In summary, our data suggest that sulfation is an important regulator of both neural precursor cell proliferation and maturation of the neural precursor cell progeny in the developing mouse spinal cord.